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Structural interrogation of enzyme mechanism and dynamics

dc.contributor.advisorStark, Holger Prof. Dr.
dc.contributor.authorMehr, Alexander
dc.date.accessioned2023-05-05T14:22:51Z
dc.date.issued2023-05-05
dc.identifier.urihttp://resolver.sub.uni-goettingen.de/purl?ediss-11858/14655
dc.identifier.urihttp://dx.doi.org/10.53846/goediss-9875
dc.format.extentXXX Seitende
dc.language.isoengde
dc.rights.urihttp://creativecommons.org/licenses/by/4.0/
dc.subject.ddc572de
dc.titleStructural interrogation of enzyme mechanism and dynamicsde
dc.typedoctoralThesisde
dc.contributor.refereeStark, Holger Prof. Dr.
dc.date.examination2023-04-18de
dc.description.abstractengThe pyruvate dehydrogenase complex (PDHc) is one of the largest macromolecular enzyme complexes, and serves an universally important metabolic function by converting pyruvate into acetyl-CoA. Therefore, the PDHc connects glycolysis with the citric acid cycle, and fatty acid biosynthesis. The protein complex consists of the three different enzymes E1, E2, and E3, which catalyze pyruvate decarboxylation, acetyl transfer, and re-oxidation of the lipoyl moiety sequentially. A small protein domain, which is attached to a flexible linker, transfers reaction intermediates to the different active sites in a process called substrate channeling. PDH deficiency leads to lethal neurological disorders, and the PDHc might be a valuable antibiotic target. Its importance for cellular function, and unique structural organization makes it an attractive target for structural investigations. Multiple crystal structures of its individual subunits have been elucidated. However, structures re-ported thus far were generally limited to 1.7-2.6 Å resolution, and little information is avail-able about the interaction with its essential cofactor lipoamide. Furthermore, structural in-formation of reaction intermediates and dynamics of the E1, a thiamine diphosphate dependent enzyme, have not been elucidated so far. In this thesis, the full decarboxylation reaction of the Bacillus subtilis PDH E1 was analyzed by time-resolved serial crystallography. Parameters such as purification, micro-crystallization, sample delivery, and data collection were optimized. This lead to the collection of datasets with high completeness and reproducible diffraction to 2.3-2.5 Å. Structures of multiple time points during the reaction were elucidated. The time points showed a structural non-equivalence of the two active sites called half-of-the-sites reactivity. A post-crystallization protocol was established, which resulted in macro-crystals diffracting to high resolution. Multiple decarboxylation intermediates were elucidated at 0.95-1.03 Å, which helped the interpretation of the time-resolved data. The pre-decarboxylation intermediate exhibited enzyme mediated substrate- and cofactor bond distortions, which might contribute to its efficient catalysis. The post-decarboxylation intermediate showed a similar tautomeric equilibrium as observed in the pyruvate oxidase, which highlights common features of thiamine dependent enzymes. The high resolution allowed the visualization of a proton network connecting both active sites. A substrate mediated protonation asymmetry was observed, which could be a driving factor in half-of-the-sites reactivity. The structures also helped the elucidation of the E1 inhibition mechanism by fluoropyruvate. The formation of a tricyclic ThDP form including a seven-membered ring was observed. High resolution structures of E2 and E3 enzymes in complex with the essential cofactor lipoamide were elucidated. These lead to the proposal of a new catalytic aspartate residue residing in a loop region, which closes its conformation upon cofactor binding. The E3-lipoamide interaction was trapped for the first time, which showed, how flavin dependent oxidoreductases accommodate the thiol moieties of their respective substrates. In summary, successful elucidation of reaction intermediates showed the complementarity of time-resolved crystallography and classical cryo-crystallography. Optimization of bio-chemical and technical protocols, such as post-crystallization, cryo-trapping, and data collection strategies, lead to the discovery of previously unobserved structural details of all three subunits of the pyruvate dehydrogenase complex in high spatial and temporal resolution.de
dc.contributor.coRefereeMata, Ricardo Prof. Dr.
dc.subject.engpyruvate dehydrogenase complexde
dc.subject.engX-ray crystallographyde
dc.subject.engTime-resolved crystallographyde
dc.subject.engThiamine diphosphatede
dc.identifier.urnurn:nbn:de:gbv:7-ediss-14655-4
dc.date.embargoed2024-04-17
dc.affiliation.instituteGöttinger Graduiertenschule für Neurowissenschaften, Biophysik und molekulare Biowissenschaften (GGNB)de
dc.subject.gokfullBiologie (PPN619462639)de
dc.description.embargoed2024-04-17de
dc.identifier.ppn1844737314
dc.notes.confirmationsentConfirmation sent 2023-05-05T14:45:01de


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